GPR30 co-localizes with cholinergic neurons in the basal forebrain and enhances potassium-stimulated acetylcholine release in the hippocampus
Introduction
Studies show that 17-ß-estradiol (E2) can significantly enhance basal forebrain cholinergic function and improve performance on a variety of cognitive tasks (reviewed in Gibbs, 2010). For example, E2 increases the expression of choline acetyltransferase (ChAT) mRNA and protein in the medial septum and nucleus basalis magnocellularis (Gibbs, 1996, McMillan et al., 1996, Bohacek et al., 2008), and increases both ChAT activity (Luine, 1985, Gibbs, 2000) and high affinity choline uptake (O’Malley et al., 1987, Singh et al., 1995) in the hippocampus and cortex. E2 also increases potassium-stimulated acetylcholine (ACh) release in the hippocampus (Gibbs et al., 1997, Gabor et al., 2003), which correlates with effects on place learning (Marriott and Korol, 2003). Effects of E2 on acquisition of T-maze and radial arm maze tasks are blocked by either cholinergic denervation of the hippocampus (Gibbs, 2007), or by the inhibition of M2 muscarinic ACh receptors (Daniel and Dohanich, 2001). In rats, E2 attenuates amnestic effects induced by scopolamine (Fader et al., 1998, Fader et al., 1999, Gibbs, 1999). A similar effect was recently reported in humans (Dumas et al., 2006). Conversely, scopolamine at appropriate doses can block the memory-enhancing effects of E2 injected directly into the hippocampus (Packard, 1998). These findings demonstrate that E2 enhances basal forebrain cholinergic function, and that the cholinergic projections play a critical role in mediating effects of E2 on cognitive performance. The mechanisms, however, by which E2 affects the cholinergic neurons remain unclear.
We hypothesize that some of the effects of E2 on basal forebrain cholinergic neurons are mediated by GPR30. GPR30 is a member of the seven transmembrane G-protein coupled receptor family and mediates rapid signaling in response to E2 in several cell lines (Thomas et al., 2005, Prossnitz et al., 2008). Previous work from our laboratory showed that treating ovariectomized rats with G-1, a selective GPR30 agonist (Bologa et al., 2006), enhanced the rate of acquisition on a delayed matching-to-position (DMP) T-maze task similar to E2 (Hammond et al., 2009). Effects of E2 on DMP acquisition rely upon cholinergic projections to the hippocampus (Gibbs, 2007). The purpose of the present study was to evaluate GPR30 expression in the rat forebrain, to determine whether cholinergic neurons in the basal forebrain express GPR30, and to test whether activation of GPR30 affects cholinergic function in the hippocampus as reflected by a change in ACh release.
Section snippets
Animals
A total of 22 young adult, ovariectomized, female Sprague–Dawley rats were purchased from Hilltop Laboratories. Ten rats were used for the immunohistochemical (n = 5) and RT-PCR (n = 5) experiments and 12 were used for in vivo microdialysis. Rats were individually housed on a 12 h day/night cycle with food and water freely available. All procedures were carried out in accordance with PHS policies on the use of animals in research, and with the approval of the University of Pittsburgh's Institutional
Characterization of GPR30 expression in the forebrain
GPR30-IR was detected in many regions of the rat forebrain including the hippocampus, septum, frontal cortex, striatum and nucleus basalis (Fig. 2). Strong staining was seen in the cytoplasm and in some processes. Some cells were more strongly labeled than others. Staining was particularly strong in presumptive cholinergic cell groups (Fig. 2B and C). Staining was greatly reduced in sections incubated with the GPR30 antibody plus 10 μg/mL C-terminal blocking peptide (Fig. 2F). Staining was not
Discussion
The data show that GPR30 is expressed in many regions of the forebrain that are important for learning and memory including the hippocampus, frontal cortex, striatum and in basal forebrain cholinergic nuclei. These findings are consistent with and augment previous studies showing significant GPR30 expression in hippocampus, and in specific regions of the hypothalamus and spinal cord (Brailoiu et al., 2007, Dun et al., 2009). Our co-localization studies show that the majority of cholinergic
Conclusions
GPR30-IR is expressed in many regions of the rat forebrain. In addition, the vast majority of cholinergic neurons in the forebrain express GPR30-IR, and we provide evidence that activation of GPR30 can enhance ACh release in the hippocampus similar to estradiol. This supports the hypothesis that GPR30 plays a role in mediating the effects of E2 on basal forebrain cholinergic function. Studies show that cholinergic inputs to the hippocampus and cerebral cortex play a critical role in mediating
Role of funding source
Funding for this study was provided by F31 AG034035-01A1 (RLH), NIH Grant R21 AG031794 (RBG) and a grant from the University of Pittsburgh Central Research Development Fund (RBG). The funding sources had no role in the collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication.
Conflict of interest
All authors declare that they do not have any conflict of interest.
Acknowledgement
The GPR30 primary antibody was a gift from Edward Filardo (Brown University).
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